Studies on the purification and characterization of the (S)-adenosyl-L-methionine sterol methyl transferase from Saccharomyces cerevisiae
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The ERG6 gene that encodes (S)-adenosyl-L- ethionme:A^'^^^^^ to A^"^^^*^ sterol methyl transferase (SMT) enzyme from Saccharomyces cerevisiae was mtroduced into plasmid pET23a(+) and the resulting native protein was overexpressed in BL21(DE3) host cells under control of a T7 promoter. This enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, anion exchange, and hydrophobic interaction chromatography. The wild type enzyme fi-om S. cerevisiae was also purified to apparent homogenehy by differential centriftigation, solubilization, anion exchange chromatography, gel permeation chromatography, and hydrophobic mteraction chromatography. A^-terminal sequence analysis of the first 10 amino acids of the purified recombmant SMT protem confirmed the identhy of the start triplet and the expected primary structure. The enzyme exhibited a turnover number of 0.01/s and an isoelectric pomt of 5.95. A combmation of Superose 6 chromatography and sodium dodecyl sulfateipolyacrylimide gel electrophoresis (SDSiPAGE), in the case of the recombinant, and Sephacryl S-300 HR and SDSrPAGE, for the wild type showed that the purified SMT enzyme fi-om both sources possessed a native molecular weight of 172,000 dahons and was tetrameric. The purified SMT enzyme generated kinetics in which velochy versus substrate curves relative to zymosterol (preferred sterol acceptor molecule) and AdoMet were sigmoidal rather than hyperbolic, indicating cooperativity between the subunits. Studies on product formation using [27- C]zymosterol and [H3-methylJAdoMet mcubated with the pure SMT enzyme confirmed the reaction mechanism of sterol methylation to involve a 1,2-hydride shift of H-24 to C-25 from the Re-face of the origmal 24,25- double bond.